The goal of this project is to develop mechanisms by which protein folding can be coupled to unfolding, in order to introduce allosteric control into proteins and enzymes. Two techniques are introduced for this purpose: mutually exclusive folding (MEF) and alternate frame folding (AFF). MEF uses the free energy stored in the folded structure of one domain to unfold another domain within the same molecule. The two domains cannot exist simultaneously in their native states; hence the molecule interconverts between two functional forms. By matching a receptor domain with an enzymatic or functional domain, one can create new molecules in which ligand binding causes a desired biological function. AFF is a novel mechanism for coupling binding to conformational change in a simple, predictable, and well-defined manner. It involves permutation of the amino acid sequence and partial sequence duplication. The resulting protein switches between native structures that are structurally similar, but possess different topologies. The driving force is provided by the universal linkage between ligand binding and protein folding. The conformational change is harnessed to a biological function or to an output signal. AFF can be implemented within a single protein or can be combined with MEF in a two- domain fusion to establish allosteric regulation. The aims will generate biosensors for calcium and sugars, protein scaffolds for development of versatile (universal) sensors, a potent anti-HIV toxin, and a method for introducing conditional functionality to a protein of choice. These molecules will establish a framework for designing a wide variety of proteins with novel molecular recognition and therapeutic properties. PUBLIC HEALTH RELEVANCE: This project develops new mechanisms for introducing allosteric control into proteins. This technology is used to create proteins of medical significance (anti-HIV therapeutics) and biological interest (biosensors for metals, sugars, and other small molecules).